G-coupled receptor showing selective affinity for ATP

ABSTRACT

The present invention relates to an isolated G-protein coupled receptor, nucleic acid sequence encoding the receptor, and host cells capable of expressing the receptor. The invention further comprises methods for detecting the expression of a G-protein coupled receptor, and methods for identifying agonists or antagonists of the receptor. The invention still further encompasses methods for preparing an isolated G-protein coupled receptor.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present invention is a continuation of U.S. application Ser.No.09/254,783, filed Aug. 16, 1999, which claims priority toPCT/BE98/00108, filed Jul. 9, 1998, which claims priority toEP97870101.9, filed Jul. 9, 1997. The contents of each priorityapplication is incorporated by reference herein in its entirety.

OBJECT OF THE PRESENT INVENTION

[0002] The present invention concerns a new G protein-coupled receptorhaving selective affinity for ATP and the nucleic acid molecule encodingsaid receptor, vectors comprising said nucleic acid molecule, cellstransformed by said vector, antibodies directed against said receptor,nucleic acid probes directed against said nucleic acid molecule,pharmaceutical compositions comprising said products and non humantransgenic animals expressing the receptor according to the invention orthe nucleic acid molecule according to said receptor.

BACKGROUND OF THE INVENTION

[0003] An impressive number of P2 receptors subtypes has been clonedsince 1993. A new molecular nomenclature has then been created in whichG protein-coupled P2 receptors have been named P2Y while P2 receptorshaving an intrinsic ion channel activity have been named P2X. The P2Yfamily encompasses selective purinoceptors (the P2Y₁ receptor activatedby ATP and ADP), nucleotide receptors responsive to both adenine anduracil nucleotides (P2Y₂ receptor: activated equipotentally by ATP andUTP) and pyrimidinoceptors (the P2Y₃ and P2Y₆ receptors activated byUDP; the P2Y₄ receptor: activated by UTP). The P2Y₅ and P2Y₇ receptorsdisplay limited homologies with the other members of the P2Y family.They have been included in this family especially on the basis ofradioligand binding studies showing affinities for adenine nucleotides(1-18).

SUMMARY OF THE INVENTION

[0004] The present invention concerns a new receptor having the aminoacids sequence of FIG. 1 or any receptor which presents more than 50%,preferably more than 70%, more preferably more than 85%, morespecifically more than 95% homology with the amino acids sequence ofFIG. 1.

[0005] The present invention concerns also the receptor having at leastthe amino acids sequence of FIG. 1 or a portion thereof, preferably anamino acids sequence wherein the large extracellular part (the NH₂portion of 450 amino acids sequence the end of which (♦) is representedon FIG. 1; note that the amino acid numbering of FIG. 1 is incorrect:i.e., residue number 437 in FIG. 1 is correctly numbered as residue 424in SEQ ID NO: 1) has been truncated or active parts of said portion suchas the sixth and seventh transmembrane domains comprising the aminoacids: His⁶⁸⁶, Arg⁶⁸⁹ and Arg⁷²⁸.

[0006] The present invention is also related to said NH₂ portion of 450amino acids sequence, including peptides reproducing or mimicking aportion of this sequence or of organic molecules sharing the effects ofthese peptides.

[0007] Indeed, the inventors have discovered that either the wholereceptor having the amino acids sequence of FIG. 1 or its portion(preferably the amino acids sequence wherein the large extracellularpart of 450 amino acids has been truncated and starting from (♦) inFIG. 1) seems to have the same industrial application (said portion willbe identified hereafter as the P2Y₁₁ receptor or sequence).

[0008] The first industrial application of this receptor or its portionsis the screening of agonists and antagonists of said receptor which mayhave advantageous pharmaceutical or diagnostical properties. The secondindustrial application of the receptor according to the invention or ofits portions or of active parts of its portions is the identification ofpatients who may present genetic disorders induced by an inactivereceptor or by an inactive portion of said receptor.

[0009] According to a preferred embodiment of the present invention,said receptor is a human receptor.

[0010] ATP seems to be the preferential natural agonist of thisreceptor: UTP, UDP, AP₄A, AP₆A, AMP and adenosine seem to be unable tostimulate the phosphoinositide pathway or were much less potent thatATP.

[0011] Therefore, the invention is also related to a new G-coupledreceptor, its portions or active parts of its portions having aselective affinity for ATP. “A selective affinity for ATP” means thatATP is able to induce the formation of a functional response (preferablythe accumulation of Inositol triphosphate IP₃ and a rise ofintracellular Ca²⁺) in a short time of incubation with said agonist(preferably in less than 5 min; more preferably less than 1 min) whilethe other known agonists of P2Y (UTP, UDP, AP₄A, AP₆A, AMP andadenosine) were unable to stimulate said receptor or were much lesspotent than ATP and induce a detectable functional response by saidreceptor.

[0012] The present invention is also related to a nucleic acid molecule,such as a DNA molecule or an RNA molecule, encoding the receptor, itsportions or active parts of its portions according to the invention.

[0013] Preferably, said DNA molecule is a cDNA molecule or a genomic DNAmolecule.

[0014] Preferably, said nucleic acid molecule has more than 50%,preferably more than 70%, more preferably more than 85%, morespecifically more than 95% homology with the DNA sequence shown in FIG.1.

[0015] Preferably, the invention is related to a nucleic acid moleculewhich has more than 50%, preferably more than 70%, more preferably morethan 85%, more specifically more than 95% homology with this DNAsequence (shown in FIG. 1), wherein the DNA sequence encoding the 450amino acids of the NH₂ portion were truncated.

[0016] The present invention is also related to the vector comprisingthe nucleic acid molecule according to the invention. Preferably, saidvector is adapted for expression in a cell and comprises the regulatoryelements necessary for expressing the amino acid molecule in said celloperatively linked to the nucleic acid sequence according to theinvention as to permit expression thereof.

[0017] Preferably, said cell is selected from the group consisting ofbacterial cells, yeast cells, insect cells or mammalian cells. Thevector according to the invention is a plasmid or a virus, preferably abaculovirus, an adenovirus or a Semliki Forest virus.

[0018] The present invention concerns also the cell transformed by thevector according to the invention, said cell is preferably non-neuronalin origin and is selected from the group consisting of a COS-7 cell, aCHO cell, an LM(tk−) cell, an NIH-3T3 cell or a 1321N1 astrocytoma cell.

[0019] The present invention is also related to a nucleic acid probecomprising the nucleic acid molecule according to the invention, of atleast 15 nucleotides capable of specifically hybridising with a uniquesequence included in the sequence of the nucleic acid molecule encodingthe receptor according to the invention. Said nucleic acid probe may bea DNA or an RNA molecule.

[0020] The invention concerns also an antisense oligonucleotide having asequence capable of specifically hybridising to an mRNA moleculeencoding the receptor according to the invention so as to preventtranslation of said mRNA molecule or an antisense oligonucleotide havinga sequence capable of specifically hybridising to the cDNA moleculeencoding the receptor according to the invention.

[0021] Said antisense oligonucleotide may comprise chemical analogs ofnucleotide or substances which inactivate mRNA, or be included in an RNAmolecule endowed with ribozyme activity.

[0022] Another aspect of the present invention concerns a ligand(preferably an antibody) other than known molecules, especially the ATP,capable of binding to the receptor according to the invention and ananti-ligand (preferably also an antibody) capable of competitivelyinhibiting the binding of said ligand to the receptor according to theinvention.

[0023] Preferably, said antibody is a monoclonal antibody directed to anepitope of the receptor according to the invention and present on thesurface of a cell expressing said receptor.

[0024] The invention concerns also the pharmaceutical compositioncomprising an effective amount of oligonucleotide according to theinvention, effective to decrease the activity of said receptor bypassing through a cell membrane and binding specifically with mRNAencoding the receptor according to the invention in the cell so as toprevent its translation. The pharmaceutical composition comprises also apharmaceutically acceptable carrier capable of passing through said cellmembrane.

[0025] Preferably, in said pharmaceutical composition, theoligonucleotide is coupled to a substance, such as a ribozyme, whichinactivates mRNA.

[0026] Preferably, the pharmaceutically acceptable carrier comprises astructure which binds to a receptor on a cell capable of being taken upby cell after binding to the structure. The structure of thepharmaceutically acceptable carrier in said pharmaceutical compositionis capable of binding to a receptor which is specific for a selectedcell type.

[0027] Preferably, said pharmaceutical composition comprises an amountof the antibody according to the invention effective to block thebinding of a ligand to the receptor according to the invention and apharmaceutically acceptable carrier.

[0028] The present invention concerns also a transgenic non human mammaloverexpressing (or expressing ectopically) the nucleic acid moleculeencoding the receptor according to the invention.

[0029] The present invention also concerns a transgenic non human mammalcomprising a homologous recombination knockout of the native receptoraccording to the invention.

[0030] According to a preferred embodiment of the invention, thetransgenic non human, mammal whose genome comprises antisense nucleicacid complementary to the nucleic acid according to the invention is soplaced as to be transcripted into antisense mRNA which is complementaryto the mRNA encoding the receptor according to the invention and whichhybridises to mRNA encoding said receptor, thereby reducing itstranslation. Preferably, the transgenic non human mammal according tothe invention comprises a nucleic acid molecule encoding the receptoraccording to the invention and comprises additionally an induciblepromoter or a tissue specific regulatory element.

[0031] Preferably, the transgenic non human mammal is a mouse.

[0032] The invention relates also to a method for determining whether aligand as an agonist or an antagonist of the receptor according to theinvention can be specifically bound to said receptor; said methodcomprising the steps of contacting a cell or a cell extract from cellstransfected with a vector according to the invention and expressing thenucleic acid molecule encoding said receptor, possibly isolating amembrane fraction from the cell extract, contacting the ligand with themembrane fraction or with the cell under conditions permitting bindingof said ligand to the receptor and detecting, possibly by means of abioassay such as a modification in the second messenger concentration ora modification in the cellular metabolism (preferably determined by theacidification rate of the culture medium), an increase in the receptoractivity, thereby determining whether the ligand binds to the receptor,possibly as an agonist or as an antagonist of said receptor.

[0033] Preferably, the second messenger assay comprises measurement ofintracellular cAMP, intracellular inositol phosphate (IP3),intracellular diacylglycerol (DAG) concentration or intracellularcalcium mobilisation.

[0034] Preferably, the cell used in said method is a mammalian cell nonneuronal in origin, such as a COS-7 cell, a CHO cell, a LM(tk−) cell anNIH-3T3 cell or 1321N1 cell. In said method, the ligand is notpreviously known.

[0035] The invention is also related to the ligand isolated and detectedby any of the preceding methods.

[0036] The present invention concerns also the pharmaceuticalcomposition which comprises an effective amount of an agonist or anantagonist of the receptor according to the invention, effective toreduce the activity of said receptor and a pharmaceutically acceptablecarrier.

[0037] The P2Y₁₁ transcripts (obtained from the nucleotidic sequencestarting from (♦) in FIG. 1) are detectable in HL-60 human leukaemiacells. Expression of P2Y₁₁ receptor mRNA is increased by agents (ripnoicacid, DMSO) known to induce the granulocytic differenciations of HL-60cells. However, the P2Y₁₁ transcripts could not be detected in matureneutrophils. Therefore, a first industrial application of the productaccording to the invention is the diagnostic of leukaemia, preferably byNorthern blot analysis using the nucleotidic sequence encoding the P2Y₁₁receptor according to the invention.

[0038] The present invention is also related to a diagnostic device orkit comprising the elements for the diagnostic of specific leukaemia,preferably HL-60 human leukaemia, comprising the receptor according tothe invention, the nucleic acid sequence encoding said receptor, anucleic acid probe comprising the nucleic acid molecule according to theinvention of at least 15 nucleotides capable of specifically hybridisingwith a unique sequence included in the sequence of the nucleic acidmolecule encoding the receptor according to the invention, such as anantisense oligonucleotide or a ligand such as an antibody, preferably amonoclonal antibody, capable of binding or competitively inhibiting thebinding of a ligand to the receptor according to the invention. Saiddiagnostic device or kit could be used for the specific diagnostic orfor the monitoring of the evolution of tumoral cells, especiallyleukaemia HL-60 cells.

[0039] Therefore, the previously described methods may be used for thescreening of drugs (having advantageously anti-tumoral properties) whichspecifically bind to the receptor according to the invention.

[0040] Another industrial application of the present invention isrelated to the use of said drugs, preferably ligands or anti-ligandsaccording to the invention, for the prevention and/or the treatment ofspecific diseases such as neutropenie or agranulocytose infections orcancer.

[0041] The invention is also related to the drugs isolated and detectedby any of these methods.

[0042] The present invention concerns also a pharmaceutical compositioncomprising said drugs and a pharmaceutically acceptable carrier.

[0043] The invention is also related to a method of detecting expressionof a receptor according to the invention by detecting the presence ofmRNA coding for a receptor, which comprises obtaining total RNA or totalmRNA from the cell and contacting the RNA or mRNA so obtained with thenucleic acid probe according to the invention under hybridisingconditions and detecting the presence of mRMA hybridised to the probe,thereby detecting the expression of the receptor by the cell.

[0044] The hybridisation conditions above-described are preferablystandard stringent conditions as described by Sambrook et al.(§9.47-9.51 in Molecular Cloning: A Laboratory Manual, Cold SpringHarbour, Laboratory Press, New York (1989)).

[0045] The present invention concerns also a method for diagnosing apredisposition to a disorder associated with the activity of thereceptor according to the invention. Said method comprises:

[0046] a) obtaining nucleic acid molecules of subjects suffering fromsaid disorder;

[0047] b) performing a restriction digest of said nucleic acid moleculeswith a panel of restriction enzymes;

[0048] c) electrophoretically separating the resulting nucleic acidfragments on a sized gel;

[0049] d) contacting the resulting gel with a nucleic acid probe capableof specifically hybridising to said nucleic acid molecule and labelledwith a detectable marker;

[0050] e) detecting labelled bands which have hybridised to the saidnucleic acid molecule labelled with a detectable marker to create aunique band pattern specific to subjects suffering from said disorder;

[0051] f) preparing nucleic acid molecules obtained for diagnosis bystep a-c; and

[0052] g) comparing the unique band pattern specific to the nucleic acidmolecule of subjects suffering from the disorder from step e and thenucleic acid molecule obtained for diagnosis from step f to determinewhether the patterns are the same or different and to diagnose therebypredisposition to the disorder if the patterns are the same.

[0053] A last aspect of the present invention concerns a method ofpreparing the receptor according to the invention, which comprises:

[0054] a) constructing a vector adapted for expression in a cell whichcomprises the regulatory elements necessary for the expression ofnucleic acid molecules in the cell operatively linked to nucleic acidmolecule encoding said receptor so as to permit expression thereof,wherein the cell is selected from the group consisting of bacterialcells, yeast cells, insect cells and mammalian cells;

[0055] b) inserting the vector of step a in a suitable host cell;

[0056] c) incubating the cell of step b under conditions allowing theexpression of the receptor according to the invention;

[0057] d) recovering the receptor so obtained; and

[0058] e) purifying the receptor so recovered, thereby preparing anisolated receptor according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0059]FIG. 1 represents the nucleotide and deduced amino acid sequenceof the new human P2Y receptor. The putative phosphorylation sites byprotein kinase C or by calmodulin-dependent protein kinases areindicated respectively by a black circle () or a black diamond (♦). Thepotential N-glycosylation site is indicated by a black square (▪).

[0060]FIG. 2 represents dendrogram of the structural relatedness of theP2Y₁₁ receptor with the other P2Y subtypes. The plot was constructedusing the multiple sequence alignment program Pileup of the GCG package.The P2Y₅-like published sequence (18) is identical to the P2Y₉ sequencesubmitted to the GenBank/EMBL Data Bank.

[0061]FIG. 3 represents Northern blot analysis of P2Y₁₁ messengerexpression. Each lane of the MTN blot contains 2 μg of polyA⁺ RNA fromseveral human tissues. Each lane of the HL-60 blot contains 10 μg oftotal RNA from differentiated or undifferentiated HL-60 cells.Hybridization with the probe was performed as described under Materialsand Methods. The pictures of the MTN II blot and the HL-60 blot wereobtained respectively, from an autoradiography and from a PhosphorlmagerSI (Molecular Dynamics). The 2 kb-length P2Y₁₁ transcripts are indicatedby a black arrow.

[0062]FIG. 4 represents concentration-action curves of severalnucleotides on IP₃ and cAMP accumulation in cells transfected with theP2Y₁₁ receptor. 1321N1 and CHO-Kl transfected cells were assayed for theaccumulation of, respectively, IP₃ (A) or cAMP (B) in response tovarious concentrations of the following nucleotides: ATP, 2MeSATP, ADPand 2MeSADP. Incubation times were 30 s for IP₃ measurements and 15 minfor cAMP assays. The data represent the means ±S.D. of triplicateexperimental points and are representative of two independentexperiments.

DESCRIPTION OF A PREFERRED EMBODIMENT OF THE PRESENT INVENTION

[0063] Experimental Procedures

[0064] Materials

[0065] Trypsin was from Flow Laboratories (Bioggio, Switzerland).Culture media, G418, fetal calf serum (FCS), restriction enzymes and Taqpolymerase were purchased from GIBCO ERL (Grand Island, N.Y.). Theradioactive products myo-D-[2-³H]inositol (17.7 Ci/mmol) and [α³²P]ATP(800 Ci/mmol) were from Amersham (Ghent, Belgium). Dowex AG1X8 (formateform) was from Bio-Rad Laboratories (Richmond, Calif.) ATP, ADP, AMP,adenosine, UTP, UDP, AP₄A, AP₆A, all-trans retinoic acid (RA) and12-O-tetradecanoylphorbol-3-acetate (TPA) were obtained from SigmaChemical Co. (St. Louis, Mo.). 2-methylthio-ATP (2MeSATP),2-methylthio-ADP (2MeSADP) and 8 (p-sulfophenyl) theophylline were fromResearch Biochemicals International (Natick, Mass.). Forskolin waspurchased from Calbiochem (Bierges, Belgium). Indomethacin and dimethylsulfoxide (DMSO) were from Merck (Netherlands). Rolipram was obtainedfrom the Laboratoires Jacques Logeais (Trappes, France). The HL-60 humancell line was obtained from the American Type Culture Collection(Rockville, USA). The human genomic DNA library was from Stratagene (LaJolla, Calif.). pEFIN3 is an expression vector obtained from Euroscreen(Brussels, Belgium). Multiple Human Tissues Northern blot (MTN) werefrom Clontech (Palo Alto, Calif.).

[0066] Cloning and Sequencing

[0067] A human placenta cDNA library was screened at moderate stringencywith an [α³²P] dATP labelled P2Y₄ receptor probe corresponding to apartial sequence covering the third to the seventh transmembranesegments. Three overlapping clones encoding a new G protein-coupledreceptor were isolated, but did not contain the 3′ end of the codingregion. A human genomic DNA library was then screened with this partialsequence to obtain the complete sequence of this new receptor. Thehybridization conditions for screening the two libraries were 6× SSC (1×SSC: 0.15 M NaCl, 0.015 M sodium citrate) and 40% formamide at 42° C.for 14 hours and the final washing conditions were 0.5× SSC, 0.1% SDS at60° C. Four genomic clones were purified and shown to contain the 3′ endof the open reading frame missing in the cDNA clones. The sequence wasobtained on both strands after subcloning of overlapping restrictionfragments in M13mp18 and M13mp19 using the Sanger dideoxy nucleotidechain termination method.

[0068] Northern Blot Analysis

[0069] Two blots of human organs (MTN I and MTN II: 2 μg polyA⁺RNA/lane) and a blot containing total RNA from differentiated andundifferentiated HL-60 cells (10 μg of total RNA/lane) were hybridizedwith a probe corresponding to the new receptor in order to characterizeits tissue distribution. The HL-60 cells were maintained in RPMI 1640supplemented with 10% FCS, 5 mM L-glutamine, 50 U/ml penicillin and 50μg/ml streptomycin at 37° C. with 5% CO₂. The HL-60 cells were incubatedduring six days with or without 1 μM retinoic acid or 1.25% DMSO orduring eight hours with 25 nM TPA. The RNA from the differentiated orundifferentiated HL-60 cells was prepared with the RNeasy kit (Quiagen).The blots were prehybridized 8 hours at 42° C. in a 50% formamide, 2%SDS solution and hybridized for 18 hours in the same solutionsupplemented with the [α³²p] labelled probe. The final washingconditions were 0.1× SSC and 0.1% SDS at 55° C. The blots were exposedduring twelve days and visualized as an autoradiography or using thePhosphorlmager SI (Molecular Dynamics).

[0070] Cell Culture and Transfection

[0071] The complete sequence of the new receptor according to theinvention was subcloned between the Hind III and Nhe I sites of thebicistronic pEFIN3 expression vector. 1321N1 and CHO-Kl cells weretransfected with the recombinant pEFIN3 plasmid or with the plasmidalone using the calcium phosphate precipitation method as described(19). The transfected cells were selected with 400 μg/ml G418 incomplete medium (10% FCS, 100 units/ml penicillin, 100 μg/mlstreptomycin and 2.5 μg/ml amphotericin B in Duibecco's modifiedEagle'medium (DMEM)) two days after transfection and maintained in thesame medium (10).

[0072] Inositol Phosphates (IP) Measurement

[0073] 1321N1 cells were labelled for 24 hours with 10 mCi/ml [³H]inositol in inositol free DMEM containing 5% FCS, antibiotics,amphotericin, sodium pyruvate and 400 μg/ml G418. Cells were washedtwice with Krebs-Ringer Hepes (KRH) buffer of the following composition(124 mM NaCl, 5 mM KCl, 1.25 mM MgSO₄, 1.45 mM CaCI₂, 1.25 mM KH₂PO₄, 25mM Hepes (pH:7.4) and 8 mM glucose) and incubated in the same medium for30 min. The cells were then challenged by various nucleotides for 30 s.The incubation was stopped by the addition of an ice cold 3% perchioricacid solution. IP were extracted and separated on Dowex columns aspreviously described (20).

[0074] Cyclic AMP Measurements

[0075] Stably transfected CHO-Kl or 1321N1 cell lines were spread onPetri dishes (150.000 cells per dish) and cultured in Ham's F12 or DMEMmedium containing 10%, FCS, antibiotics, amphotericin, sodium pyruvateand 400 μg/ml G418. Cells were preincubated for 30 min in KRH bufferwith Rolipram (25 μM) and incubated for different times in the presenceof the agonists (15 min in most experiments). The incubation was stoppedby the addition of 1 ml HCl 0.1 M. The incubation medium was dried up,resuspended in water and diluted as required. Cyclic AMP was quantifiedby radioimmunoassay after acetylation as previously described (21).

[0076] Results

[0077] Cloning and Sequencing

[0078] A human cDNA placenta library was screened at moderate stringencywith a human P2Y₄ probe. Nine clones which hybridized weakly with theP2Y₄ probe were obtained, purified and analyzed. Six of themcorresponded to the sequence of the P2Y₆ receptor (10) while threeoverlapping clones corresponded to a partial sequence encoding a new Gprotein-coupled receptor, displaying about 30% identity with the otherP2Y receptors. The partial open reading frame started with an ATG-codonin a Kozak consensus but the 3′ end was missing in all three cDNAclones. The Inventors screened a human genomic DNA library using thispartial sequence as a probe. Four overlapping genomic clones wereobtained. Mapping of the coding sequence and partial sequencing allowedto determine that the gene encoding the new receptor contains an introninterrupting the coding sequence at the 5′ end of the gene. This intronseparates the three first codons from the rest of the coding sequence.Beside these first codons, the four genomic clones contained thecomplete open reading frame including the 3′ end missing in the cDNAclones. The full open reading frame appeared as 1113 base pairs (bp)long and encoded a protein of 371 amino acids containing one potentialsite for N-linked glycosylation and two potential sites forphosphorylation by protein kinase C or calmodulin-dependent proteinkinases (FIG. 1). The new receptor, provisionally named P2Y₁₁, displayssignificant homologies with the other P2Y receptors (FIG. 2). Inparticular, 33% and 28% amino acid identity were observed respectivelywith the human P2Y₁ and P2Y₂ receptors.

[0079] Tissue Distribution of the New Receptor

[0080] The tissue distribution of the new receptor transcripts wasinvestigated by Northern blotting (FIG. 3) by using a probecorresponding to a partial sequence encoding transmembrane segments 3 to7. The strongest signal was observed for human spleen and correspondedto a 2 kilobase (kb)-length messenger RNA (MTN II). A weaker signal wasobserved in small intestine (MTN II). All the lanes in MTN I (heart,brain, placenta, lung, liver, skeletal muscle, kidney, pancreas) werenegative. The Inventors also detected specific 2 kb-length transcriptsin HL-60 cells. The signal was very weak in the undifferentiated HL-60cells but increased when the cells had been treated with retinoic acidor DMSO. No increase was observed when the HL-60 cells were stimulatedwith TPA. A weak non-specific hybridization with 18S mRNA was observed.These data were confirmed with a non-overlapping probe corresponding tothe first 300 bp of the coding region, presenting limited homologieswith the other P2Y subtypes.

[0081] Functional Expression of the New Receptor in 1321N1 AstrocytomaCells

[0082] The complete sequence of the new receptor was introduced in thepEFIN3 expression vector in order to transfect the 1321N1 astrocytomacell line, used previously to characterize several P2Y subtypes (6, 10,12). The pool of G418-resistant clones was tested for its functionalresponse to several nucleotides. ATP (100 μM) induced a strong inositoltrisphosphate (IP₃) accumulation in cells transfected with therecombinant plasmid, whereas ADP, AMP, adenosine, UTP, UDP, AP₄A andAP₆A were inactive at the same concentration. All nucleotides weretotally inactive on the cells transfected with the vector alone. We thentested ATP, 2MeSATP, ADP and 2MeSADP in a large range of concentrations.As shown in FIG. 4A, ATP was the most potent agonist (EC₅₀ATP=38±7 μM;EC₅₀ 2MeSATP=118±15 μM; means ±range of two independent experiments).The effect of ADP and 2MeSADP were minimal. Pertussis toxin (50 ng/ml;24 h pretreatment) had no effect on the ATP response, whereas a lowerconcentration of pertussis toxin was previously shown to abolish theresponse to UTP in P2Y₄ transfected 1321N1 astrocytoma cells (22). Aresponse to ATP (10 μM) was also obtained following [Ca²⁺]₁ measurementsperformed on the 1321N1 transfected cells while ADP was inactive at thisconcentration.

[0083] Functional Expression of the New Receptor in CHO-Kl cells

[0084] The 1321N1 cells transfected with the new receptor displayed astrong cAMP increase in response to ATP. A much lower but significantendogeneous response due to the degradation of adenine nucleotides intoadenosine was also obtained in the 1321N1 cells transfected with thevector alone. The CHO-Kl cells express an endogeneous P2Y₂ receptorcoupled to the phosphoinositide pathway (23) but do not possessadenosine receptors coupled to adenylyl cyclase. We therefore usedCHO-Kl cells in order to characterize the coupling of the new receptorto the cAMP pathway. A pool of G418 resistant CHO-Kl clones was firsttested for its response to several nucleotides at a concentration of 100μM. ATP was able to induce a strong increase in the cAMP content,whereas it was inactive on cells transfected with the vector alone. ADP,AMP, adenosine, UTP and UDP were completely inactive.Concentration-action curves were established for ATP, 2MeSATP, ADP and2MeSADP (FIG. 4B). The rank order of potency was the same as in theinositol phosphate study on 1321N1 cells. The curves were obtained after15 min of stimulation by the agonists; however a significant cAMPresponse to ATP was already obtained after 2 min of stimulation. Theresponse to ATP (30 μM) was inhibited neither by indomethacin (10 μg/ml,present from 30 minutes before the stimulation and readded in thestimulation medium) nor by 8 (p-sulfophenyl) theophylline (100 μM).

[0085] The receptor according to the invention presents some structuralpeculiarities which differentiate it from some other P2Y subtypes.Concerning its gene structure, the coding sequence is interrupted by anintron. Comparison between the cDNA and the genomic DNA sequences hasclearly demonstrated the absence of intron in the coding region of thehuman P2Y₁ receptor (24, 25), the rat P2Y₂ receptor (26) and the ratP2Y₆ receptor (11). In terms of protein structure, the second and thirdextracellular loops are significantly longer than those of the other P2Yreceptors. The homology with the other subtypes is relatively weak(about 30%). The closest G-coupled receptor is the human P2Y₁ receptor(33%) which is also a receptor responsive to adenine nucleotides (3, 4).Mutagenesis experiments with the P2Y₂ receptor have identified threepositively charged amino acids in the sixth and seventh transmembranedomains (His²⁶², Arg²⁶⁵ and Arg²⁹²), which play a crucial role innucleotide binding (presumably by neutralizing the negative charge ofthe phosphate groups) (27). These three residues are conserved in thisnew receptor.

[0086] So far, eight P2Y receptor subtypes are described in theliterature (P2Y₁-22Y₈). In addition, two sequences related to the P2Y₅receptor and named P2Y₉ and P2Y₁₀, have been recently submitted to theGenBank/EMBL Data Bank. The P2Y₉ sequence is identical to that recentlypublished under the name “P2Y₅-like” (18). Therefore the new receptordescribed in this paper might be called P2Y₁₁. However, it is alreadyclear that the nomenclature needs a revision. It was recentlydemonstrated that the P2Y₇ receptor is actually a receptor forleukotriene B₄ (16) and there is no functional evidence that the P2Y₅and related receptors (P2Y₅-like or P2Y₉, P2Y₁₀) are nucleotidereceptors (17, 18).

[0087] Among the sixteen human organs tested by Northern blotting, P2Y₁₁transcripts of 2 kb size were only detectable in spleen, and with lowerintensity in small intestine. This distribution is reminiscent of thatof the human P2Y₆ 1.7 kb-messenger. The observation of the expression ofthe P2Y₁₁ receptor in the HL-60 cell line shows that this expression wasstrongly increased following treatment by DMSO or retinoic acid, twoagents known to induce the differentiation of these cells intogranulocytes (28). On the contrary, TPA, which is known to induce themonocytic differentiation of the HL-60 cells (29), did not stimulate theexpression of the P2Y₁₁, receptor. The confirmation of these data with asecond probe of the P2Y₁₁ cDNA, that shares little similarity with otherP2Y sequences, excludes possible cross-hybridization with another P2Yreceptor transcript. In view of the Northern blots results, it istempting to speculate that the P2Y₁₁ receptor is involved in therecently described accumulation of CAMP in ATP-stimulated HL-60 cells(30).

[0088] Among the P2Y receptors, the P2Y₁₁ subtype has the uniqueproperty to activate both the phosphoinositide and the cAMP pathways.Other cloned P2Y receptors are coupled to phospholipase C exclusively.The rank order of potency of agonists was the same for the two pathways.ATP was clearly much more potent than ADP. This difference may be evenunderestimated as a result of low level ATP contamination in ADPpreparation or conversion of ADP into ATP during assays (4, 11). On theother hand, 2MeSATP had the same maximal effect than ATP but presented alower potency, while 2MeSADP, a potent activator of the P2Y₁ and P_(2T)subtypes (4), was almost inactive. The EC₅₀ values were comparable tothose obtained in the study concerning the effects of extracellularnucleotides on the cAMP accumulation in the HL-60 cells (30).

[0089] Stimulatory effects of adenine nucleotides on the cAMP pathwayhave been described in different cell types (31, 32). In most cases, thestimulatory effect of nucleotides was inhibited by xanthines. These datasuffer from the fact that it is difficult to exclude that the effect ofadenine nucleotides is mediated by their degradation into adenosine dueto the ubiquitous presence of ectonucleotidases expressed at the cellsurface. The cAMP study has been performed with CHO-Kl cells to avoidthe endogeneous cAMP response to adenosine in the astrocytoma cell line.Neither in untransfected CHO-Kl cells nor in P2Y₁₁-transfected CHO-Klcells did adenosine increase cAMP accumulation. Furthermore the cAMPresponse to ATP was insensitive to xanthine inhibition. It was alsoinsensitive to indomethacin, indicating that is not mediated by therelease of prostaglandins. It is unlikely that the cAMP response wouldbe an indirect consequence of the calcium response since the use of ATP,which activates the phosphoinositide pathway by the activation of P2Y₂endogeneous receptors, or the use of calcium ionophores in the CHO-Klcells failed to stimulate cAMP accumulation (33). Therefore these dataconstitute the first strong evidence that a P₂ receptor can be coupledto the stimulation of adenylyl cyclase.

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1 2 1 795 PRT Homo sapiens 1 Met Gly Gln Ser Gly Arg Ser Arg His Gln LysArg Ala Arg Ala Gln 1 5 10 15 Ala Gln Leu Arg Asn Leu Glu Ala Tyr AlaAla Asn Pro His Ser Phe 20 25 30 Val Phe Thr Arg Gly Cys Thr Gly Arg AsnIle Arg Gln Leu Ser Leu 35 40 45 Asp Val Arg Arg Val Met Glu Pro Leu ThrAla Ser Arg Leu Gln Val 50 55 60 Arg Lys Lys Asn Ser Leu Lys Asp Cys ValAla Val Ala Gly Pro Leu 65 70 75 80 Gly Val Thr His Phe Leu Ile Leu SerLys Thr Glu Thr Asn Val Tyr 85 90 95 Phe Lys Leu Met Arg Leu Pro Gly GlyPro Thr Leu Thr Phe Gln Val 100 105 110 Lys Lys Tyr Ser Leu Val Arg AspVal Val Ser Ser Leu Arg Arg His 115 120 125 Arg Met His Glu Gln Gln PheAla His Pro Pro Leu Leu Val Leu Asn 130 135 140 Ser Phe Gly Pro His GlyMet His Val Lys Leu Met Ala Thr Met Phe 145 150 155 160 Gln Asn Leu PhePro Ser Ile Asn Val His Lys Val Asn Leu Asn Thr 165 170 175 Ile Lys ArgCys Leu Leu Ile Asp Tyr Asn Pro Asp Ser Gln Glu Leu 180 185 190 Asp PheArg His Tyr Ser Ile Lys Val Val Pro Val Gly Ala Ser Arg 195 200 205 GlyMet Lys Lys Leu Leu Gln Glu Lys Phe Pro Asn Met Ser Arg Leu 210 215 220Gln Asp Ile Ser Glu Leu Leu Ala Thr Gly Ala Gly Leu Ser Glu Ser 225 230235 240 Glu Ala Glu Pro Asp Gly Asp His Asn Ile Thr Glu Leu Pro Gln Ala245 250 255 Val Ala Gly Arg Gly Asn Met Arg Ala Gln Gln Ser Ala Val ArgLeu 260 265 270 Thr Glu Ile Gly Pro Arg Met Thr Leu Gln Leu Ile Lys ValGln Glu 275 280 285 Gly Val Gly Glu Gly Lys Val Met Phe His Ser Phe ValSer Lys Thr 290 295 300 Glu Glu Glu Leu Gln Ala Ile Leu Glu Ala Lys GluLys Lys Leu Arg 305 310 315 320 Leu Lys Ala Gln Arg Gln Ala Gln Gln AlaGln Asn Tyr Gln Arg Lys 325 330 335 Gln Glu Gln Arg Glu Ala His Arg LysLys Ser Leu Glu Gly Met Lys 340 345 350 Lys Ala Arg Val Gly Gly Ser AspGlu Glu Ala Ser Gly Ile Pro Ser 355 360 365 Arg Thr Ala Ser Leu Glu LeuGly Glu Asp Asp Asp Glu Gln Glu Asp 370 375 380 Asp Asp Ile Glu Tyr PheCys Gln Ala Val Gly Glu Ala Pro Ser Glu 385 390 395 400 Asp Leu Phe ProGlu Ala Lys Gln Lys Arg Leu Ala Lys Ser Pro Gly 405 410 415 Arg Lys ArgLys Arg Trp Glu Met Asp Arg Gly Ala Lys Ser Cys Pro 420 425 430 Ala AsnPhe Leu Ala Ala Ala Asp Asp Lys Leu Ser Gly Phe Gln Gly 435 440 445 AspPhe Leu Trp Pro Ile Leu Val Val Glu Phe Leu Val Ala Val Ala 450 455 460Ser Asn Gly Leu Ala Leu Tyr Arg Phe Ser Ile Arg Lys Gln Arg Pro 465 470475 480 Trp His Pro Ala Val Val Phe Ser Val Gln Leu Ala Val Ser Asp Leu485 490 495 Leu Cys Ala Leu Thr Leu Pro Pro Leu Ala Ala Tyr Leu Tyr ProPro 500 505 510 Lys His Trp Arg Tyr Gly Glu Ala Ala Cys Arg Leu Glu ArgPhe Leu 515 520 525 Phe Thr Cys Asn Leu Leu Gly Ser Val Ile Phe Ile ThrCys Ile Ser 530 535 540 Leu Asn Arg Tyr Leu Gly Ile Val His Pro Phe PheAla Arg Ser His 545 550 555 560 Leu Arg Pro Lys His Ala Trp Ala Val SerAla Ala Gly Trp Val Leu 565 570 575 Ala Ala Leu Leu Ala Met Pro Thr LeuSer Phe Ser His Leu Lys Arg 580 585 590 Pro Pro Gln Gln Gly Ala Gly AsnCys Ser Val Ala Arg Pro Glu Ala 595 600 605 Cys Ile Lys Cys Leu Gly ThrAla Asp His Gly Leu Ala Ala Tyr Arg 610 615 620 Ala Tyr Ser Leu Val LeuAla Gly Leu Gly Cys Gly Leu Pro Leu Leu 625 630 635 640 Leu Thr Leu AlaAla Tyr Gly Ala Leu Gly Arg Ala Val Leu Arg Ser 645 650 655 Pro Gly MetThr Val Ala Glu Lys Leu Arg Val Ala Ala Leu Val Ala 660 665 670 Ser GlyVal Ala Leu Tyr Ala Ser Ser Tyr Val Pro Tyr His Ile Met 675 680 685 ArgVal Leu Asn Val Asp Ala Arg Arg Arg Trp Ser Thr Arg Cys Pro 690 695 700Ser Phe Ala Asp Ile Ala Gln Ala Thr Ala Ala Leu Glu Leu Gly Pro 705 710715 720 Tyr Val Gly Tyr Gln Val Met Arg Gly Leu Met Pro Leu Ala Phe Cys725 730 735 Val His Pro Leu Leu Tyr Met Ala Ala Val Pro Ser Leu Gly CysCys 740 745 750 Cys Arg His Cys Pro Gly Tyr Arg Asp Ser Trp Asn Pro GluAsp Ala 755 760 765 Lys Ser Thr Gly Gln Ala Leu Pro Leu Asn Ala Thr AlaAla Pro Lys 770 775 780 Pro Ser Glu Pro Gln Ser Arg Glu Leu Ser Gln 785790 795 2 2427 DNA Homo sapiens 2 gaattcggca cgaggaggcc tcgtggaggacacagcagca tgggacagtc agggaggtcc 60 cggcaccaga agcgcgcccg cgcccaggcgcagctccgca acctcgaggc ctatgccgcg 120 aacccgcact cgttcgtgtt cacgcgaggctgcacgggtc gcaacatccg gcagctcagc 180 ctggacgtgc ggcgggtcat ggagccgctcactgccagcc gtctgcaggt tcgtaagaag 240 aactcgctga aggactgcgt ggcagtggctgggcccctcg gggtcacaca ctttctgatc 300 ctgagcaaaa cagagaccaa tgtctactttaagctgatgc gcctcccagg aggccccacc 360 ttgaccttcc aggtgaagaa gtactcgctggtgcgtgatg tggtctcctc actgcgccgg 420 caccgcatgc acgagcagca gtttgcccacccacccctcc tggtactcaa cagctttggc 480 ccccatggta tgcatgtgaa gctcatggccaccatgttcc agaacctgtt cccctccatc 540 aacgtgcaca aggtgaacct gaacaccatcaagcgctgcc tcctcatcga ctacaacccc 600 gactcccagg agctggactt ccgccactatagcatcaaag ttgttcctgt gggcgcgagt 660 cgcgggatga agaagctgct ccaggagaagttccccaaca tgagccgcct gcaggacatc 720 agcgagctgc tggccacggg cgcggggctgtcggagagcg aggcagagcc tgacggcgac 780 cacaacatca cagagctgcc tcaggctgtcgctggccgtg gcaacatgcg ggcccagcag 840 agtgcagtgc ggctcaccga gatcggcccgcggatgacac tgcagctcat caaggtccag 900 gagggcgtcg gggagggcaa agtgatgttccacagttttg tgagcaagac ggaggaggag 960 ctgcaggcca tcctggaagc caaggagaagaagctgcggc tgaaggcgca gaggcaggcc 1020 cagcaggccc agaatgtgca gcgcaagcaggagcagcggg aggcccacag aaagaagagc 1080 ctggagggca tgaagaaggc acgggtcgggggtagtgatg aagaggcctc tgggatccct 1140 tcaaggacgg cgagcctgga gttgggtgaggacgatgatg aacaggaaga tgatgacatc 1200 gagtatttct gccaggcggt gggcgaggcgcccagtgagg acctgttccc cgaggccaag 1260 cagaaacggc ttgccaagtc tccagggcggaagcggaagc ggtgggaaat ggatcgaggt 1320 gccaagtcct gccctgccaa cttcttggcagctgccgacg acaaactcag tgggttccag 1380 ggggacttcc tgtggcccat actggtggttgagttcctgg tggccgtggc cagcaatggc 1440 ctggccctgt accgcttcag catccggaagcagcgcccat ggcaccccgc cgtggtcttc 1500 tctgtccagc tggcagtcag cgacctgctctgcgctctga cgctgccccc gctggccgcc 1560 tacctctatc cccccaagca ctggcgctatggggaggccg cgtgccgcct ggagcgcttc 1620 ctcttcacct gcaacctgct gggcagcgtcatcttcatca cctgcatcag cctcaaccgc 1680 tacctgggca tcgtgcaccc cttcttcgcccgaagccacc tgcgacccaa gcacgcctgg 1740 gccgtgagcg ctgccggctg ggtcctggccgccctgctgg ccatgcccac actcagcttc 1800 tcccacctga agaggccgcc gcagcagggggcgggcaact gcagcgtggc caggcccgag 1860 gcctgcatca agtgtctggg gacagcagaccacgggctgg cggcctacag agcgtatagc 1920 ctggtgctgg cggggttggg ctgcggcctgccgctgctgc tcacgctggc agcctacggc 1980 gccctcgggc gggccgtgct acgcagcccaggcatgactg tggccgagaa gctgcgtgtg 2040 gcagcgttgg tggccagtgg tgtggccctctacgccagct cctatgtgcc ctaccacatc 2100 atgcgggtgc tcaacgtgga tgctcggcggcgctggagca cccgctgccc gagctttgca 2160 gacatagccc aggccacagc agccctggagctggggccct acgtgggcta ccaggtgatg 2220 cggggcctca tgcccctggc cttctgtgtccaccctctac tctacatggc cgcagtgccc 2280 agcctgggct gctgctgccg acactgccccggctacaggg acagctggaa cccagaggac 2340 gccaagagca ctggccaagc cctgcccctcaatgccacag ccgcccctaa accgtcagag 2400 ccccagtccc gtgagctgag ccaatga 2427

1. An isolated receptor comprising an amino acid sequence that is atleast 95% identical to amino acid residues 424-808 of SEQ ID NO: 1 2.Receptor according to claim 1 having a selective affinity ADP.
 3. Anisolated nucleic acid molecule encoding the receptor of claim
 1. 4.Nucleic acid molecule according to claim 3, wherein the nucleic acidmolecule is DNA or RNA molecule.
 5. DNA molecule according to claim 3,which is a cDNA molecule or a genomic DNA molecule.
 6. An isolatednucleic acid molecule having at least 95% identity to a nucleic acidmolecule consisting of nucleic acid residues 1309-2424 of the nucleicacid sequence of SEQ ID NO:2.
 7. Vector comprising the nucleic acidmolecule according to any of the claims 3 or
 6. 8. Cell comprising thevector according to claim
 7. 9. An isolated nucleic acid probecomprising a sequence of at least 15 nucleotides capable of specificallyhybridizing with a unique sequence included within the polynucleotidesequence of claim 3 or
 6. 10. An antisense oligonucleotide having asequence capable of specifically hybridizing to the polynucleotide ofclaim 3 or 7, wherein said polynucleotide is an mRNA, wherein saidantisense polynucleotide prevents translation of the mRNA molecule
 11. Apharmaceutical composition comprising an amount of the antisenseoligonucleotide according to claim 10, effective to decrease activity ofthe receptor of claim 1, or a variant, homolog or fragment thereof,wherein said antisense oligonucleotide is capable of passing through acell membrane and binding specifically with mRNA encoding said receptorin the cell so as to prevent its translation, and a pharmaceuticallyacceptable carrier capable of passing through a cell membrane.
 12. Atransgenic non human mammal comprising the polynucleotide according toclaim 3 or
 7. 13. A method of detecting the expression of the receptorhaving an amino acid sequence that is at least 95% identical to aminoacid residues 424-808 of SEQ ID NO: 1, or a variant, homologue orfragment thereof, by detecting the presence of mRNA coding saidreceptor, which comprises obtaining total RNA or total mRNA from thecell and contacting the RNA or mRNA so obtained with the nucleic acidprobe according to claim 9 under hybridizing conditions, and detectingthe presence of mRNA hybridized to the probe.
 14. A method foridentifying an antagonist or an agonist of the receptor which has anamino acid sequence that is at least 95% identical to amino acidresidues 424-808 of SEQ ID NO: 1, or a variant, homolog, or fragmentthereof, capable of alleviating an abnormality in a subject wherein theabnormality is alleviated by increasing or decreasing the activity ofthe receptor, said method comprising: (a) administering the antagonistor the agonist to a transgenic non human mammal according to claim 12,and (b) determining whether the antagonist or the agonist alleviates ordecreases the physical and behavioral abnormalities displayed by thetransgenic non human mammal as a result of receptor activity.
 15. Anantagonist or agonist identified by the method of claim
 14. 16. Apharmaceutical composition comprising an antagonist or an agonistaccording to claim 15 and a pharmaceutically acceptable carrier.
 17. Amethod of preparing the isolated receptor according to claim 1, whichcomprises: (a) constructing a vector adapted for expression in a cellwhich comprises the regulatory elements necessary for the expression ofnucleic acid molecules in the cell operatively linked to nucleic acidmolecule encoding said receptor so as to permit expression thereof; (b)inserting the vector of step a in a suitable host cell; (c) incubatingthe cell of step b under conditions allowing the expression of thereceptor according to the invention; (d) recovering the receptor soobtained; and (e) purifying the receptor so recovered.
 18. The method ofclaim 17, wherein the cell is selected from the group consisting ofbacterial cells, yeast cells, insect cells and mammalian cells.